U.S. patent application number 11/993906 was filed with the patent office on 2010-07-15 for rotatable perfused time varying electromagnetic force bioreactor and method of using the same.
This patent application is currently assigned to REGENETECH, INC.. Invention is credited to Thomas J. Goodwin, Clayton R. Parker.
Application Number | 20100178680 11/993906 |
Document ID | / |
Family ID | 42319347 |
Filed Date | 2010-07-15 |
United States Patent
Application |
20100178680 |
Kind Code |
A1 |
Goodwin; Thomas J. ; et
al. |
July 15, 2010 |
ROTATABLE PERFUSED TIME VARYING ELECTROMAGNETIC FORCE BIOREACTOR
AND METHOD OF USING THE SAME
Abstract
A rotatable perfused time varying electromagnetic force
bioreactor with a rotatable perfusable culture chamber and a time
varying electromagnetic force source operatively connected to the
rotatable perfusable culture chamber. In use, the rotatable
perfused time varying electromagnetic force bioreactor supplies a
time varying electromagnetic force to the rotatable perfusable
culture chamber of the rotatable perfused time varying
electromagnetic force bioreactor to expand cells contained
therein.
Inventors: |
Goodwin; Thomas J.; (Kemah,
TX) ; Parker; Clayton R.; (Safety Harbor,
FL) |
Correspondence
Address: |
LADAS & PARRY LLP
224 SOUTH MICHIGAN AVENUE, SUITE 1600
CHICAGO
IL
60604
US
|
Assignee: |
REGENETECH, INC.
Texas
TX
NASA
Houston
TX
|
Family ID: |
42319347 |
Appl. No.: |
11/993906 |
Filed: |
June 26, 2006 |
PCT Filed: |
June 26, 2006 |
PCT NO: |
PCT/US06/24806 |
371 Date: |
January 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11169385 |
Jun 29, 2005 |
7456019 |
|
|
11993906 |
|
|
|
|
60584572 |
Jun 30, 2004 |
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Current U.S.
Class: |
435/173.8 ;
435/289.1 |
Current CPC
Class: |
C12M 35/02 20130101;
C12M 27/10 20130101; C12N 5/0062 20130101; C12M 29/10 20130101;
C12N 13/00 20130101; C12N 2529/00 20130101; C12N 5/00 20130101 |
Class at
Publication: |
435/173.8 ;
435/289.1 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12M 1/00 20060101 C12M001/00 |
Claims
1. A rotatable perfused time varying electromagnetic force
bioreactor comprising: a rotatable perfusable culture chamber; and
a time varying electromagnetic force source comprising a coil,
operatively connected to the rotatable perfusable culture
chamber.
2. The rotatable perfused time varying electromagnetic force
bioreactor as in claim 1, wherein the time varying electromagnetic
force source is integral with the rotatable perfused time varying
electromagnetic force bioreactor.
3. The rotatable perfused time varying electromagnetic force
bioreactor as in claim 1, wherein the time varying electromagnetic
force source is adjacent to the rotatable perfused time varying
electromagnetic force bioreactor.
4. A method for producing time varying electromagnetic
force-expanded cells in a rotatable perfused time varying
electromagnetic force bioreactor, having a rotatable perfusable
culture chamber and a time varying electromagnetic force-source
comprising a coil, comprising the steps of: a. filling the
rotatable perfusable culture chamber with a culture medium; b.
placing cells in the rotatable perfusable culture chamber and
initiating a three-dimensional time varying electromagnetic force
culture; c. rotating the rotatable perfusable culture chamber about
an axis at a rotation speed; d. controlling the rotation of the
rotatable perfusable culture chamber by adjusting the rotation
speed while maintaining the three-dimensional time varying
electromagnetic force culture; and e. exposing the cells to a time
varying electromagnetic force to expand the cells.
5. The method of claim 4, wherein the time varying electromagnetic
force is in the form of a square wave.
6. The method of claim 5, wherein the time varying electromagnetic
force is of from about 0.05 gauss to about 6 gauss.
7. The method of claim 5, wherein the time varying electromagnetic
force is of from about 0.05 gauss to about 0.5 gauss.
8. The method of claim 5, wherein the square wave has a frequency
of from about 2 to about 25 cycles/second.
9. The method of claim 5, wherein the square wave has a frequency
of from about 5 to about 20 cycles/second.
10. The method of claim 5, wherein the square wave has a frequency
of 10 cycles/second.
11. The method of claim 4, wherein the three-dimensional time
varying electromagnetic force culture has the properties of
collocation of the culture medium and the cells, essentially no
relative motion of the culture medium with respect to the rotatable
perfusable culture chamber, and freedom for a three-dimensional
spatial orientation of the cells.
12. The method of claim 4, wherein the three-dimensional time
varying electromagnetic force culture is maintained by
perfusion.
13. The method of claim 4, wherein the culture medium is enriched
by a culture medium flow loop comprising a supply manifold, a pump,
an oxygenator, a rotatable perfusable culture chamber, and a
waste.
14. The method of claim 4, wherein prior to step c, the culture
medium flow loop is turned on.
15. The method of claim 13, wherein the culture medium flow loop
enriches the culture medium with at least one selected from the
group consisting of growth factors, cytokines, hormones, oxygen,
nutrients, acids, bases, buffers, and fresh culture medium prior to
entering the rotatable perfusable culture chamber.
16. The method of claim 4 wherein the rotatable perfused time
varying electromagnetic force-bioreactor is located in unit
gravity.
17. The method of claim 4 wherein the rotatable perfused time
varying electromagnetic force-bioreactor is located in
microgravity.
18. The method of claim 4, wherein the rotatable perfused time
varying electromagnetic force-bioreactor is located in less than
unit gravity.
19. The method of claim 4, wherein the culture medium further
comprises at least one cell attachment substrate.
20. The method of claim 4 wherein the rotation speed is of from
about 5 to about 120 RPM.
21. The method of claim 4 wherein the rotation speed is 10 RPM.
22. The method of claim 4, wherein the cells maintain essentially
the same three-dimensional geometry, and cell-to-cell support and
geometry as that of the cells found in vivo.
23. The method of claim 4, wherein the time varying electromagnetic
force is in the form of a differentiated square wave.
24. The method of claim 4, wherein the time varying electromagnetic
force is in the form of a delta wave.
25. The method of claim 4, wherein the cells are expanded to more
than seven times their original number.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to a rotatable
perfused time varying electromagnetic force bioreactor and a method
for using the same, and more particularly to a method of expanding
cells in a rotatable perfused time varying electromagnetic force
bioreactor while at the same time subjecting them to a time varying
electromagnetic force.
BACKGROUND OF THE INVENTION
[0002] Cell culture processes have been developed for the growth of
single cell bacteria, yeast and molds which are resistant to
environmental stresses or are encased with a tough cell wall.
Mammalian cell culture, however, is much more complex because
mammalian cells are more delicate and have more complex nutrient
and other environmental requirements in order to maintain viability
and cell growth. Large-scale cultures of bacterial type cells are
highly developed and such culture processes are less demanding and
are not as difficult to cultivate as mammalian cells. Bacterial
cells can be grown in large volumes of liquid medium and can be
vigorously agitated without any significant damage. Mammalian
cells, on the other hand, cannot withstand excessive turbulent
action without damage to the cells and are typically provided with
a complex nutrient medium to support growth.
[0003] In addition, mammalian cells have other special
requirements; in particular most animal cells typically prefer to
attach themselves to some substrate surface to remain viable and to
duplicate. On a small scale, mammalian cells have been grown in
containers with small microwells to provide surface anchors for the
cells. However, cell culture processes for mammalian cells in such
microwell containers generally do not provide sufficient surface
area to grow mammalian cells on a sufficiently large scale basis
for many commercial or research applications. To provide greater
surface areas, microcarrier beads have been developed for providing
increased surface areas for the cultured cells to attach.
Microcarrier beads with attached cultured cells require agitation
in a conventional bioreactor chamber to provide suspension of the
cells, distribution of fresh nutrients, and removal of metabolic
waste products. To obtain agitation, such bioreactor chambers have
used internal propellers or movable mechanical agitation devices
which are motor driven so that the moving parts within a chamber
cause agitation in the fluid medium for the suspension of the
microcarrier beads and attached cells. Agitation the fluid medium
may also agitate mammalian cells therein, however, subjecting them
to high degrees of fluid shear stress that can damage the cells and
limit ordered assembly of these cells according to cell derived
energy. These fluid shear stresses arise, for instance, when the
fluid media has significant relative motion with respect to chamber
walls, internal propellers or movable mechanical agitation devices,
or other chamber components. Cells may also be damaged in
bioreactor chambers with internal moving parts if the cells or
beads with cells attached collide with one another or chamber
components.
[0004] In addition to the drawbacks of cell damage, bioreactors and
other methods of culturing mammalian cells are also very limited in
their ability to provide conditions that allow cells to assemble
into tissues that simulate the spatial three-dimensional form of
actual tissues in an intact organism and at the same time allow
cells to multiply at a rate of at least seven times within seven
days. Conventional tissue culture processes limit, for similar
reasons, the capacity for cultured tissues to, for instance,
develop a highly functionally specialized or differentiated state
considered crucial for mammalian cell differentiation and secretion
of specialized biologically active molecules of research and
pharmaceutical interest. Unlike microorganisms, the cells of higher
organisms such as mammals form themselves into high order
multicellular tissues. Although the exact mechanisms of this
self-assembly are not known, in the cases that have been studied
thus far, development of cells into tissues has been found to be
dependent on orientation of the cells with respect to each other
(the same or different type of cell) or other anchorage substrate
and/or the presence or absence of certain substances (factors) such
as hormones. In summary, no conventional culture process is capable
of simultaneously achieving sufficiently low fluid shear stress,
sufficient three-dimensional spatial freedom, and for sufficiently
long periods for critical cell interactions (with each other or
substrates) to allow excellent modeling of in vivo cell and tissue
structure, and at the same time, provides accelerated expansion,
growth in the size of tissue and/or tissue constructs and/or growth
in the number of cells, while maintaining the cell or tissue three
dimensional geometry, and cell-to-cell geometry and support.
[0005] For example, U.S. Pat. No. 5,155,035, Wolf et al., provides
a method for culturing tissues, tissue constructs, and cells
utilizing a perfused bioreactor that overcomes prior problems
without subjecting the tissue and/or cells to destructive amounts
of shear. The Wolf et al. disclosure, however, provides for a very
low rate of production. In fact, the Wolf et al. device, and method
of using the same, provides an insufficiently low production rate
such that it is not of substantial commercial value.
[0006] It is highly desirable, therefore, to have a rotatable
perfused time varying electromagnetic force bioreactor that has a
rotatable perfusable culture chamber and a time varying
electromagnetic force ("TVEMF")-source operatively connected to the
rotatable perfusable culture chamber. It is also highly desirable
to have a method for expanding cells using a rotatable perfused
TVEMF-bioreactor that, when in use, not only achieves sufficiently
low fluid shear stress, sufficient three-dimensional spatial
freedom, and for sufficiently long periods for critical cell
interactions (with each other or substrates), but at the same time,
provides accelerated expansion while maintaining the cell or tissue
three dimensional geometry, and cell-to-cell geometry and
support.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a rotatable perfused
TVEMF-bioreactor comprising a rotatable perfusable culture chamber
and a TVEMF source operatively connected to the rotatable
perfusable culture chamber.
[0008] The present invention also relates to a method for expanding
cells in a rotatable perfused TVEMF-bioreactor comprising the steps
of filling a rotatable perfusable culture chamber of the rotatable
perfused TVEMF-bioreactor with a culture medium, placing cells in
the rotatable perfusable culture chamber to initiate a
three-dimensional TVEMF culture, rotating the rotatable perfusable
culture chamber about an axis at a rotation speed, controlling the
rotation of the rotatable perfusable culture chamber by adjusting
the rotation speed to maintain the three-dimensional TVEMF culture,
and exposing the cells to a TVEMF. The TVEMF is preferrably in the
form of a delta wave, more preferably a differentiated square wave,
and most preferably a square wave (following a Fourier curve).
Preferably the method of the present invention has the properties
of collocation of the culture medium and the cells, essentially no
relative motion of the culture medium with respect to the rotatable
perfusable culture chamber, and freedom for three-dimensional
spatial orientation of the cells, while at the same time,
maintaining essentially the same three-dimensional geometry, and
cell-to-cell support and geometry as that of the cells found in
vivo.
[0009] Other aspects, features, and advantages of the present
invention will be apparent from the following description of the
preferred embodiments of the invention given for the purpose of
disclosure. This invention may be more fully described by the
preferred embodiment(s) as hereinafter described, but is not
intended to be limited thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] In the drawings,
[0011] FIG. 1 schematically illustrates a preferred embodiment of a
culture medium flow loop;
[0012] FIG. 2 is a vertical cross-sectional view of a preferred
embodiment of a rotatable perfused TVEMF-bioreactor;
[0013] FIG. 3 is a cross section along line 3-3 of FIG. 2;
[0014] FIG. 4 is a vertical cross sectional view of a preferred
embodiment of a rotatable perfused TVEMF-bioreactor;
[0015] FIG. 5 is a vertical cross sectional view of a rotatable
perfused TVEMF-bioreactor;
[0016] FIG. 6 is the orbital path of a typical cell in a
non-rotating reference frame.
[0017] FIG. 7 is a graph of the magnitude of deviation of a cell
per revolution.
[0018] FIG. 8 is a representative cell path as observed in a
rotating reference frame of the culture medium.
[0019] FIG. 9 is a side view of a TVEMF-device.
[0020] FIG. 10 is an elevated front view of the TVEMF-device shown
in FIG. 9.
[0021] FIG. 11 is an elevated front view of the TVEMF-device also
shown in FIGS. 9 and 10 further showing a rotatable perfused TVEMF
bioreactor adjacently located therein.
DETAILED DESCRIPTION OF THE DRAWINGS
[0022] In the simplest terms, a rotatable perfused TVEMF-bioreactor
comprises a rotatable perfusable culture chamber and a time varying
electromagnetic force source ("TVEMF-source") operatively connected
to the rotatable perfusable culture chamber. Preferred rotatable
perfused TVEMF-bioreactors are described herein. FIGS. 2 and 3
illustrate a preferred embodiment of a rotatable perfused
TVEMF-bioreactor 10 with an adjacent TVEMF-source (not shown). FIG.
4 is a cross section of a rotatable perfused TVEMF-bioreactor 10
for use in the present invention in a preferred form with an
integral TVEMF-source. FIG. 5 illustrates a rotatable perfused
TVEMF-bioreactor 10 with an integral TVEMF-source. FIGS. 9-11 show
an adjacent TVEMF-source of a rotatable perfused
TVEMF-bioreactor.
[0023] When in use, the present invention provides for the support
of respiratory gas exchange in, supply of nutrients in, and removal
of metabolic waste products from a three-dimensional TVEMF culture
by the perfusion of culture medium through the rotatable perfusable
culture chamber. The term "perfusable," and similar terms, is
intended to mean that the culture medium may be poured over,
diffused or permeated through, cells in a perfusable rotatable
culture chamber of a rotatable perfused TVEMF-bioreactor. The
perfusion may preferably be through a culture medium flow loop as
illustrated in FIG. 1, more preferably via direct injection, and
most preferably via exchange across a diffusion membrane. In the
drawings, referring now to FIG. 1, illustrated is a preferred
embodiment of a culture medium flow loop 1 of a rotatable perfused
TVEMF-bioreactor having a rotatable perfusable culture chamber 19,
an oxygenator 21, an apparatus for facilitating the directional
flow of the culture medium, preferably by the use of a main pump
15, and a supply manifold 17 for the selective input of culture
medium requirements such as, but not limited to, nutrients 6,
buffers 5, fresh medium 7, cytokines 9, growth factors 11, and
hormones 13. In this preferred embodiment, the main pump 15
provides fresh medium from the supply manifold 17 to the oxygenator
21 where the medium is oxygenated and passed through the rotatable
perfusable culture chamber 19. The waste in the spent medium from
the rotatable perfusable culture chamber 19 is removed, preferably
by the main pump 15, and delivered to the waste 18 and the
remaining cell culture medium not removed to the waste 18 is
returned to the manifold 17 where it may preferably receive a fresh
charge of culture medium requirements before recycling by the pump
15 through the oxygenator 21 to the rotatable perfusable culture
chamber 19.
[0024] In operation, the culture medium is circulated through the
three-dimensional TVEMF culture in the rotatable perfusable culture
chamber 19 and preferably around the culture medium flow loop 1, as
shown in FIG. 1. In this preferred embodiment of a culture medium
flow loop 1, adjustments are made in response to chemical sensors
(not shown) that maintain constant conditions within the rotatable
perfusable culture chamber 19. Controlling carbon dioxide pressures
and introducing acids or bases corrects pH. Oxygen, nitrogen, and
carbon dioxide are dissolved in a gas exchange system (not shown)
in order to support cell respiration. The culture medium flow loop
1 adds oxygen and removes carbon dioxide from a circulating gas
capacitance. Although FIG. 1 is one preferred embodiment of a
culture medium flow loop that may be used in the present invention,
the invention is not intended to be so limited. The input of
culture medium requirements such as, but not limited to, oxygen,
nutrients, buffers, fresh medium, cytokines, growth factors, and
hormones into a rotatable perfused TVEMF-bioreactor can also be
performed manually, automatically, or by other control means, as
can be the control and removal of waste and carbon dioxide.
[0025] FIG. 2 is a cross-sectional side view of a preferred
embodiment of a rotatable perfused TVEMF-bioreactor 10 that is
operatively connected to an adjacent TVEMF-source (not shown)
having an input end 12 and an output end 14. In FIG. 2, an outer
tubular housing 20 rotatably supported for rotation about a
horizontal central axis 21 and about an input shaft 23 and an
output shaft 25 which are aligned with the central axis 21 (not
shown except by dashed line). The outer tubular housing 20 has an
interior wall 27, preferably cylindrically shaped, an output
transverse end wall 28, and an input traverse end wall 29 that
generally define a rotatable perfusable culture chamber 30,
preferably cylindrically shaped, preferably elongated. A spur gear
32 is attached to one end of the outer tubular housing 20 and is
driven by a motor 33 to rotate the housing 20 about its horizontal
central axis 21.
[0026] Coaxially disposed about the central axis 21 is an inner
filter assembly 35, preferably tubular, that is rotatably mounted
on the input shaft 23 and is coupled (as shown by the dashed line
36) to the output shaft 25. The output shaft 25, in turn, is
rotatably supported in an output stationary housing 40 and the
output shaft 25 has an externally located output spur gear 41 that
is connected to a first independent drive motor 42 for rotating the
output shaft 25 and the inner filter assembly 35 independently of
the outer housing 20. The annular space between the inner filter
assembly 35 and the interior wall 27 of the outer tubular housing
20 define the rotatable perfusable culture chamber 30 located about
the horizontal axis 21. Intermediate of the outer wall 43 of the
inner filter assembly 35 and the interior wall 27 of the outer
housing 20 is an intermediate blade member system 50 which
preferably includes two lengthwise extending blade members 50a and
50b which are preferably equiangularly spaced from one another
about the central axis 21. Each of the blade members 50a and 50b at
a first longitudinal end 51 has a second radial arm 52 which is
rotatably supported on the output shaft 25 and at a second
longitudinal end 54 has a second radial arm 55 which is coupled to
the input shaft 23 (shown by dashed line 56). The input shaft 23,
in turn, is rotatably mounted in an input stationary housing 60 and
the input shaft 23 has an input spur gear 61 that is driven by a
second independent drive motor 62 for rotation of the blade member
system 50 independent of the rotation of the outer housing 20.
[0027] As shown in FIG. 3, the angular rotation of the three
sub-assemblies 20, 35 and 50, i.e., the inner filter assembly or
member 35, the outer housing 20, and the intermediate blade member
system 50, may preferably be at the same angular rate and in the
same direction about a horizontal rotational axis and preferably
substantially in the same direction about a horizontal axis so that
there is substantially no relative movement between the three
sub-assemblies. This condition of operation obtains a clinostat
suspension of microcarrier beads in a fluid medium within the
rotatable perfusable culture chamber 30 of the rotatable perfused
TVEMF-bioreactor 10 without turbulence.
[0028] The rotation of the filter 35 can preferably be started and
stopped to, for instance, add culture medium, which will cause
turbulence on the surface of the filter 35 and keep the surface
clean. The blade members 50a and 50b assist cells as they grow to
maintain spatial positions in the rotating culture medium. This is
particularly helpful for higher density cells, tissues, and
tissue-like structures, for instance, bone cells. By rotating the
fluid and the outer housing 20, the velocity gradient at the wall
boundary layer is nearly eliminated.
[0029] The rotatable perfused TVEMF-bioreactor 10 of FIG. 2, in
operation provides for culture medium preferably containing fresh
nutrients and gases to be input to an input passageway 66 in the
input stationary housing 60 and connects to an input longitudinal
passageway 67 in the input shaft 23 by virtue of a sealed input
rotative coupling 70. The input passageway 67 in the input shaft 23
couples to a radial supply passageway 72 in an end cap of the outer
housing 20 by virtue of a sealed input rotative coupling 75. The
radial supply passageway 72, in turn, connects to space apart a
radially directed input end input passage 78 and output end input
passage 79 in the outer housing 20 where the input end input
passage 78, and output end input passage 79 are located at opposite
ends of the rotatable perfusable culture chamber 30. As shown by
the arrows, when medium is input at both ends of the rotatable
perfusable culture chamber 30, the medium moves radially outward
toward the interior wall 27 of the outer housing 20 and then moves
longitudinally in a horizontal direction toward a midpoint plane
generally indicated by a vertically dashed line 80 and then moves
radially inwardly toward the outer wall 43 of inner filter assembly
35. Thus the medium in the chamber 30 has a generally toroidal type
of motion in radial planes on either side of the midpoint plane 80
of the outer housing 20. The inner filter assembly 35 has apertures
82 along its length for exit of culture medium from the rotatable
perfusable culture chamber 30 to the interior and, while not
illustrated in FIG. 2, preferably there is a lengthwise extending
filter cloth located across the apertures 82 that prevents
microcarrier bead members in the chamber 30 from exiting through
the apertures 82. Spent culture medium in the rotatable perfusable
culture chamber 30 thus is passed to the interior 85 of the inner
filter assembly 35 and exits via an output longitudinal passageway
86 in the output shaft 25 to an output rotative coupling output 88
in the output stationary housing 40 and to an output passageway 89
preferably to the return of the culture medium flow loop for
recharging (not shown).
[0030] Turning now to the preferred embodiment of a rotatable
perfused TVEMF-bioreactor 10 comprising a rotatable perfusable
culture chamber 230 and a TVEMF-source operatively connected to the
rotatable perfusable culture chamber 230, wherein the TVEMF-source
is an annular wire heater 296. The annular wire heater 296 is
integral with the rotatable perfusable culture chamber 230. The
rotatable perfusable culture chamber 230 is preferably transparent,
and further comprising an outer housing 220 which includes a first
290 and second 291 transverse end cap member, preferably
cylindrically shaped, having facing first 228 and second 229 end
surfaces arranged to receive an inner member 293, preferably
cylindrical tubular, preferably transparent, and more preferably
glass, and an outer tubular member 294, preferably transparent, and
preferably glass. Suitable pressure seals are well known in the art
and are preferably provided. Between the inner 293 and outer 294
tubular members is an annular wire heater 296 that can preferably
be utilized for obtaining the proper incubation temperatures for
TVEMF-expansion. The wire heater 296 can also preferably be used as
a TVEMF-source that, in use, supplies a TVEMF to the rotatable
perfusable culture chamber 230. The first end cap member 290 and
second end cap member 291 have inner curved surfaces adjoining the
end surfaces 228, 229 for promoting smoother flow of the mixture
within the culture chamber 230. The first end cap member 290, and
second end cap member 291 have a first central fluid transfer
journal member 292 and second central fluid transfer journal member
295, respectively, that are rotatably received respectively on an
input shaft 223 and an output shaft 225. Each transfer journal
member 294, 295 has a flange to seat in a recessed counter bore in
an end cap member 290, 291 and is attached by a first lock washer
and ring 297, and second lock washer and ring 298 against
longitudinal motion relative to a shaft 223, 225. Each journal
member 294, 295 has an intermediate annular recess that is
connected to longitudinally extending, circumferentially arranged
passages. Each annular recess in a journal member 292, 295 is
coupled by a first radially disposed passage 278 and second
radially disposed passage 279 in an end cap member 290 and 291,
respectively, to first input coupling 203 and second input coupling
204. In operation, culture medium in a radial passage 278 or 279
flows through a first annular recess and the longitudinal passages
in a journal member 292 or 295 to permit access to the medium
through a journal member 292, 295 to each end of the journal member
292, 295 where preferably the access is circumferential about a
shaft 223, 225.
[0031] Attached to the end cap members 290 and 291 are a first
tubular bearing housing 205, and second tubular bearing housing 206
containing ball bearings which relatively support the outer housing
220 on the input 223 and output 225 shafts. The first bearing
housing 205 has an attached first sprocket gear 210 for providing a
rotative drive for the outer housing 220 in a rotative direction
about the input 223 and output 225 shafts and the longitudinal axis
221. The first bearing housing 205, and second bearing housing 206
also preferably have provisions for electrical take out of the
annular wire heater 296 and preferably any other sensor, for
instance a sensor to detect a change in the location of the cells,
preferably and/or a sensor to detect a change in the pH and/or the
temperature of the three-dimensional TVEMF culture.
[0032] The inner filter assembly 235 includes inner 215 and outer
216 tubular members having perforations and/or apertures along
their lengths and have a first 217 and second 218 inner filter
assembly end cap member with perforations. The inner tubular member
215 is preferably constructed in two pieces with an interlocking
centrally located coupling section and each piece attached to an
end cap 217 or 218. The outer tubular member 216 is preferably
mounted between the first 217 and second inner filter assembly end
caps.
[0033] The end cap members 217, 218 are respectively rotatably
supported on the input shaft 223 and the output shaft 225. The
inner member 215 is rotatively attached to the output shaft 225
preferably by a pin and an interfitting groove 219. A cloth 224,
preferably nylon, with a weave, preferably ten-microns, is disposed
over the outer surface of the outer member 216 and is attached at
either end, preferably with O-rings. Because the inner member 215
is attached to a slot in the output drive shaft 225, preferably by
a coupling pin, the output drive shaft 225 can rotate the inner
member 215. The inner member 215 is coupled by the first 217 and
second 218 end caps that support the outer member 216. The output
shaft 225 is extended through bearings in a first stationary
housing 240 and is coupled to a first sprocket gear 241. As
illustrated, the output shaft 225 has a tubular bore 222 that
extends from a first passageway 289 in the first stationary housing
240 located between seals to the inner member 215 so that, in use,
a flow of culture medium can be exited from the inner member 215
through the stationary housing 240.
[0034] Between the first 217 and second 218 end caps for the inner
member 235 and the journals 292, 295 in the outer housing 220, are
a first 227 and second 226 hub for the blade members 250a and 250b.
The second hub 226 on the input shaft 223 is coupled to the input
shaft 223 by a pin 231 so that the second hub 226 preferably
rotates with the input shaft 223. Each hub 227, 226 has axially
extending passageways for the transmittal of culture medium through
a hub.
[0035] The input shaft 223 extends through bearings in the second
stationary housing 260 for rotatable support of the input shaft
223. A second longitudinal passageway 267 extends through the input
shaft 223 to a location intermediate of retaining washers and rings
that are disposed in a second annular recess 232 between the
faceplate and the housing 260. A third radial passageway 272 in the
second end cap member 291 permits culture medium in the recess to
exit from the second end cap member 291. While not shown, the third
passageway 272 connects to each of the passages 278 and 279,
preferably through piping and a Y joint.
[0036] A sample port is shown in FIG. 4, where a first bore 237
extending along a first axis intersects a corner 233 of the chamber
230 and forms a restricted aperture 234. Preferably the bore 237
has a counter bore and a threaded ring at one end to receive a
cylindrical valve member 236, preferably threadedly. The valve
member 236 protrudes slightly into the interior of the chamber 230,
and the valve member 236 comprises a complimentarily formed tip to
engage the opening 234 and. Preferably, an O-ring 243 on the valve
member 236 provides a seal. A second bore 244 along a second axis
intersects the first bore 237 at a location between the O-ring 243
and the opening 234. Preferably an elastomer or plastic stopper 245
closes the second bore 244 that can be entered with a syringe for
removing a sample. To remove a sample, the valve member 236 is
backed off to access the opening 234 and the bore 244. A syringe
can then be used to extract a sample and the opening 234 can be
reclosed, and therefore, no outside contamination reaches the
interior of the TVEMF-bioreactor 10.
[0037] In operation, culture medium is input to the second port or
passageway 266 to the shaft passageway and thence to the first
radially disposed 278 and second radially disposed passageways 279
via the third radial passageway 272. In operation, when the culture
medium enters the chamber 230 via the longitudinal passages in the
journals 292, 294 the culture medium impinges on an end surface
228, 229 of the hubs 227, 226 and is dispersed radially as well as
axially through the passageways in the hubs 227, 226. Culture
medium passing through the hubs 227, 226 impinges on the end cap
members 217, 218 and is dispersed radially. The flow of entry
culture medium is thus radially outward away from the longitudinal
axis 221 and flows in a toroidal fashion from each end to exit
through the polyester cloth 224 and openings in the filter assembly
235 to exit via the passageways 266 and 289. By controlling the
rotational speed and direction of rotation of the outer housing
220, chamber 230, and inner filter assembly 235 any desired type of
culture medium action can be obtained. Preferably a substantially
clinostat operation can be obtained together with a continuous
supply of culture medium.
[0038] FIG. 5 is a cross-sectional elevated side view of the
preferred embodiment of a rotatable perfused TVEMF bioreactor 10.
The preferred embodiment of a rotatable perfused TVEMF-bioreactor
10, illustrated in FIG. 5, shows a wire coil 144, which is a
TVEMF-source, that is integral with the rotatable perfused
TVEMF-bioreactor 10, but is separate from the annular wire heater
296, both of which can be used as a TVEMF-source. The preferred
embodiment of a rotatable perfused TVEMF bioreactor 10 illustrated
in FIG. 5 is different from the preferred embodiment of a rotatable
perfused TVEMF-bioreactor illustrated in FIG. 4 because FIG. 4 only
discloses an annular wire heater 296 that can preferably be used as
a TVEMF-source.
[0039] If a TVEMF is not applied using an integral TVEMF-source for
instance an annular wire heater 296, as in FIG. 4, or an integral
TVEMF-source for instance a wire coil 144 as in FIG. 5, it can be
supplied by another preferred TVEMF-source. For instance, FIGS.
9-11 illustrate a preferred embodiment of another TVEMF-source, a
TVEMF-device 140 that may preferably supply a TVEMF to a
three-dimensional TVEMF culture in a rotatable perfused
TVEMF-bioreactor which does not have an integral TVEMF-source, for
example the rotatable perfused TVEMF-bioreactor illustrated in FIG.
2. Specifically, FIG. 9 is a preferred embodiment of a TVEMF-device
140 that may be operatively connected to a rotatable perfusable
culture chamber. FIG. 9 is an elevated side perspective of the
TVEMF-device 140 which comprises a support base 145, a coil support
146 disposed on the base 145 with a wire coil 147 wrapped around
the support 146. FIG. 10 is an elevated front perspective of a
TVEMF-device 140 illustrated in FIG. 9. FIG. 11 illustrates the
TVEMF-device operatively connected to a rotatable perfused
TVEMF-bioreactor 148, such as that of FIG. 2, so that the rotatable
perfused TVEMF-bioreactor 148 in FIG. 11 has an adjacent
TVEMF-source. In use, a rotatable perfused TVEMF-bioreactor 148 may
preferably be inserted into the coil support 146 which is disposed
on a support base 145 and which is wound by a wire coil 147. Since
the TVEMF-device 140 is adjacent to the rotatable perfused
TVEMF-bioreactor 148, the TVEMF-device 140 can be reused as a
TVEMF-source. In addition, since the TVEMF-device 140 is adjacent
to the rotatable perfused TVEMF-bioreactor 148, the TVEMF-device
140 can be used to generate a TVEMF in all types of
TVEMF-bioreactors, preferably rotatable.
[0040] As various changes could be made in rotatable perfused
TVEMF-bioreactors subjected to a time varying electromagnetic force
as are contemplated in the present invention, without departing
from the scope of the invention, it is intended that all matter
contained herein be interpreted as illustrative and not
limiting.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The following definitions are meant to aid in the
description and understanding of the defined terms in the context
of the present invention. The definitions are not meant to limit
these terms to less than is described throughout this application.
Furthermore, several definitions are included relating to
TVEMF--all of the definitions in this regard should be considered
to complement each other, and not construed against each other.
[0042] As used throughout this application, the term "TVEMF" refers
to "time varying electromagnetic force". As discussed above, the
TVEMF of this invention is in a delta wave, more preferably a
differential square wave, and most preferably a square wave
(following a Fourier curve). Preferably, the square wave has a
frequency of about 2 to about 25 cycles/second, more preferably
about 5 to about 20 cycles/second, and for example about 10
cycles/second, and the TVEMF-source has an RMS value preferably of
from about 0.1 mA to about 1000 mA, more preferably about 1 mA to
about 10 mA, for instance 6 mA. The TVEMF applied to a rotatable
perfused TVEMF-bioreactor is preferably in the range of from about
0.05 to about 6.0 gauss, more preferably of from about 0.05 to
about 0.5 gauss, and most preferably about 0.5 gauss. However,
these parameters are not meant to be limiting to the TVEMF of the
present invention, as such may vary based on other aspects of this
invention. TVEMF may be measured for instance by standard equipment
such as an EN131 Cell Sensor Gauss Meter.
[0043] As used throughout this application, the term "rotatable
perfused TVEMF-bioreactor" is meant to comprise a TVEMF-source
operatively connected to a rotatable perfusable culture chamber. In
use, a rotatable perfused TVEMF-bioreactor refers to a bioreactor
to which TVEMF is applied, that may be rotated and that provides a
method for sustaining the three-dimensional TVEMF culture by
perfusion, as described for instance in the Description of the
Drawings, above. In operation, the TVEMF, at an appropriate gauss
level, is delivered to the interior portion of the rotatable
perfusable culture chamber. The volume of the rotatable perfusable
culture chamber is preferably of from about 100 ml to about 3
liters. See for instance FIGS. 2, 3, 4 and 5 herein for examples
(not meant to be limiting) of a rotatable perfused TVEMF-bioreactor
of the present invention. Preferably, a rotatable perfused
TVEMF-bioreactor allows for the exchange of growth medium
(preferably with additives) and for oxygenation of the
three-dimensional TVEMF culture. Without being bound by theory, the
rotatable perfused TVEMF-bioreactor provides for the
TVEMF-expansion of cells for several days or more.
[0044] As used throughout this application, the term
"three-dimensional TVEMF culture" and similar terms, refers to a
mixture of cells with culture medium that allows the cells to
expand, for TVEMF-expansion of the cells in a rotatable perfusable
culture chamber. The culture medium and the cells, tissue, or
tissue-like structure in combination is referred to as a
three-dimensional TVEMF culture when located in the rotatable
perfusable culture chamber of the rotatable perfused
TVEMF-bioreactor, and/or after TVEMF has been delivered thereto.
The three-dimensional TVEMF-culture is preferably supported by
TVEMF-expansion in a rotatable perfused TVEMF-bioreactor wherein
cells maintain their three-dimensional geometry, and cell-to-cell
support and geometry. Three-dimensional tissue and/or tissue-like
structures can also develop from the cells and be sustained and
further expanded in the three-dimensional TVEMF culture.
[0045] As used throughout this application, the term "operatively
connected," and similar terms, is intended to mean that the
TVEMF-source can be connected to the rotatable perfusable culture
chamber in a manner such that when in operation, the TVEMF-source
can impart a TVEMF to the rotatable perfusable culture chamber and
the cells, tissue, or tissue-like structures contained therein. The
TVEMF-source may be operatively connected if it is integral with
the rotatable perfusable culture chamber, and may also be
operatively connected if it is adjacently located to the rotatable
perfusable culture chamber. The TVEMF-source is operatively
connected if it can impart a TVEMF to the inside of the rotatable
perfusable culture chamber.
[0046] As used throughout this application, the term "exposing,"
and similar terms, refers to the process of supplying a TVEMF to
cells contained in a rotatable perfusable culture chamber. In
operation, the present invention provides that the TVEMF-source is
turned on and set at a preferred gauss range and a preferred wave
form so that the same is delivered via the TVEMF-source, preferably
a wire coil, more preferably an annular wire heater, most
preferably a TVEMF-device. The TVEMF is then delivered through the
TVEMF-source to the cells in the rotatable perfusable culture
chamber thus exposing the cells to the TVEMF.
[0047] As used throughout this application, the term "cells" refers
to a cell in any form, for example, individual cells, tissue, cell
aggregates, cells pre-attached to cell attachment substrates for
instance microcarrier beads, tissue-like structures, or intact
tissue resections. The cells may be from, but are not limited to,
the following sources: mammalian, reptilian, avian, from fish, from
yeast, and bacterial.
[0048] As used throughout this application, the term "culture
medium" and similar terms, refers to a liquid comprising, but not
limited to, culture media and nutrients, which is meant for the
sustenance of cells over time. The culture medium may be enriched
with any of the following, but is not limited thereto: culture
media, buffer, growth factors, hormones, and cytokines. The culture
medium is supplied to the cells for suspension therein and to
support TVEMF-expansion. The culture medium may preferably be mixed
with the cells before it is added to the rotatable perfusable
culture chamber of the rotatable perfused TVEMF-bioreactor, or may
preferably be added before the cells are added to the rotatable
perfusable culture chamber thereby mixing the culture medium and
the cells in the rotatable perfusable culture chamber. The culture
medium and the cells combination is referred to as a
three-dimensional TVEMF culture when located in the rotatable
perfused TVEMF-bioreactor, and/or after TVEMF has been delivered
thereto. The culture medium can preferably be enriched and/or
refreshed during TVEMF-expansion as needed. Waste contained in
culture medium, as well as culture medium itself, can be removed
from the three-dimensional TVEMF culture as needed. Waste contained
in the spent culture medium can be, but is not limited to,
metabolic waste, dead cells, and other toxic debris. The culture
medium can preferably be enriched with oxygen and preferably has
oxygen, carbon dioxide, and nitrogen carrying capabilities.
[0049] As used throughout this applications, the term, "placing,"
and similar terms, refers to the process of suspending cells in
culture medium before adding the combination cells and culture
medium to the rotatable perfusable culture chamber of the rotatable
perfused TVEMF-bioreactor. The term "placing," may also refer to
adding cells to culture medium that is already present in the
rotatable perfusable culture chamber. The cells may be suspended in
additional preferred liquids including, for instance, PBS and/or
plasma. The culture medium and the cells combination is referred to
as a three-dimensional TVEMF culture when located in the rotatable
perfused TVEMF-bioreactor, during and after TVEMF has been
delivered thereto. Cells can be placed into the rotatable
perfusable culture chamber along with cell attachment substrates
and also with
[0050] As used throughout this application, the term
"TVEMF-expansion," and similar terms, refers to the process of
increasing the number of cells in a rotatable perfused
TVEMF-bioreactor, by subjecting the cells to a TVEMF of from about
0.05 to about 6.0 gauss. Preferably the cells are TVEMF-expanded to
more than seven times their original number. The expansion of cells
in a rotatable perfused TVEMF-bioreactor according to the present
invention provides for cells that maintain, or have the same or
essentially the same, three-dimensional geometry and cell-to-cell
support and cell-to-cell geometry as the cells prior to
TVEMF-expansion. Other aspects of TVEMF-expansion may also provide
the exceptional characteristics of the cells of the present
invention. Not to be bound by theory, TVEMF-expansion not only
provides for high concentrations of cells that maintain their
three-dimensional geometry and cell-to-cell support, but also
supports and maintains the growth of three-dimensional tissues and
tissue-like structures.
[0051] As used throughout this application, the term "cell-to-cell
geometry" refers to the geometry of cells including the spacing,
distance between, and physical relationship of the cells relative
to one another. For instance, TVEMF-expanded cells, including those
of tissues, cell aggregates, and tissue-like structures, of this
invention stay in relation to each other as in the body. The
expanded cells are within the bounds of natural spacing between
cells, in contrast to for instance two-dimensional expansion
containers, where such spacing is not preserved over time and
TVEMF-expansion.
[0052] As used throughout this application, the term "cell-to-cell
support" refers to the support one cell provides to an adjacent
cell. For instance, tissues, cell aggregates, tissue-like
structures, and cells maintain interactions such as chemical,
hormonal, neural (where applicable/appropriate) with other cells in
the body. In the present invention, these interactions are
maintained within normal functioning parameters, meaning they do
not for instance begin to send toxic or damaging signals to other
cells (unless such would be done in the natural cellular and tissue
environment).
[0053] As used throughout this application, the term
"three-dimensional geometry" refers to the geometry of cells in a
three-dimensional state (same as or very similar to their natural
state), as opposed to two-dimensional geometry for instance as
found in cells grown in a Petri dish, where the cells become
flattened and/or stretched. Not to be bound by theory, but the
three-dimensional geometry of the cells is maintained, supported,
and preserved such that the cell can develop into three-dimensional
cell aggregates, tissues and/or tissue-like structures in the
three-dimensional TVEMF culture of the rotatable perfused
TVEMF-bioreactor. Furthermore, tissues can also be cultured in the
rotatable perfused TVEMF-bioreactor, while at the same time,
maintaining the three-dimensional geometry, and cell-to-cell
support and geometry.
[0054] For each of the above three definitions, relating to
maintenance of "cell-to-cell support" and "cell-to-cell geometry"
and "three-dimensional geometry" of the cells of the present
invention, the term "essentially the same" means that natural
geometry and support are provided in TVEMF-expansion, so that the
cells are not changed in such a way as to be for instance
dysfunctional, toxic or harmful to other cells.
[0055] In operation, cells are placed into the rotatable perfusable
culture chamber of the rotatable perfused TVEMF-bioreactor. The
rotatable perfusable culture chamber is rotated over a period of
time during which a TVEMF is generated in the rotatable perfusable
culture chamber by the TVEMF-source. By "during which," it is
intended that the initiation of the delivery of the TVEMF may be
before, concurrent with, or after rotation of the rotatable
perfusable culture chamber is initiated. Upon completion of the
period of time, the TVEMF-expanded cells are removed from the
rotatable perfusable culture chamber. In a more complex rotatable
perfused TVEMF-bioreactor, a culture medium enriched with culture
medium requirements preferably including, but not limited to, cell
culture media, buffer, nutrients, hormones, cytokines, and growth
factors, which provides sustenance to the cells, can be
periodically refreshed and removed.
[0056] In use, the present invention provides a stabilized culture
environment into which cells may be introduced, suspended,
assembled, grown, and maintained with improved retention of
delicate three-dimensional structural integrity by simultaneously
minimizing the fluid shear stress, providing three-dimensional
freedom for cell and substrate spatial orientation, and increasing
localization of cells in a particular spatial region for the
duration of the culture. In use, the present invention provides
these three criteria (hereinafter referred to as "the three
criteria above"), and at the same time, has a TVEMF-source that
exposes the cells to a TVEMF supplied by a TVEMF-source, preferably
in a square wave, more preferably a differential square wave, most
preferably a delta wave, to the cells so that TVEMF-expansion of
the cells is enhanced. Of particular interest to the present
invention is the dimension of the rotatable perfusable culture
chamber, the sedimentation rate of the cells, the rotation rate,
the external gravitational field, and the TVEMF.
[0057] The stabilized culture environment referred to in the
operation of present invention is that condition in the culture
medium, particularly the fluid velocity gradients, prior to
introduction of cells, which will support a nearly uniform
suspension of cells upon their introduction thereby creating a
three-dimensional TVEMF culture upon addition of the cells. In a
preferred embodiment, the culture medium is initially stabilized
into a near solid body horizontal rotation about an axis within the
confines of a similarly rotating chamber wall of a rotatable
perfused TVEMF-bioreactor. The chamber walls are set in motion
relative to the culture medium and internal chamber components so
as to initially introduce essentially no fluid stress shear field
therein. Cells are introduced to, and move through, the culture
medium in the stabilized culture environment and a TVEMF is applied
thereto thus creating a three-dimensional TVEMF culture. The cells
move under the influence of gravity, centrifugal, and coriolus
forces, and the presence of cells within the culture medium of the
three-dimensional TVEMF culture induces secondary effects to the
culture medium. The significant motion of the culture medium with
respect to the rotatable perfusable culture chamber walls,
significant fluid shear stress, and other fluid motions, is due to
the presence of these cells within the culture medium.
[0058] In most cases the cells with which the stabilized culture
environment is primed sediment at a slow rate preferably under 0.1
centimeter per second. It is therefore possible, at this early
stage of the three-dimensional TVEMF culture, to select from a
broad range of rotational rates (preferably of from about 5 to
about 120 RPM) and chamber diameters (preferably of from about 0.5
to about 36 inches). Preferably, the slowest rotational rate is
advantageous because it minimizes equipment wear and other
logistics associated with handling of the three-dimensional TVEMF
culture.
[0059] Not to be bound by theory, rotation about a substantially
horizontal axis with respect to the external gravity vector at an
angular rate optimizes the orbital path of cells suspended within
the three-dimensional TVEMF culture. In operation, the cells expand
to form a mass of cell aggregates, three-dimensional tissues,
and/or tissue-like structures, which increase in size as the
three-dimensional TVEMF culture progresses. The progress of the
three-dimensional TVEMF culture is preferably assessed by a visual,
manual, or automatic determination of an increase in the diameter
of the three-dimensional tissue mass in the three-dimensional TVEMF
culture. An increase in the size of the cell aggregate, tissue, or
tissue-like structure in the three-dimensional TVEMF culture may
require appropriate adjustment of rotational rates in order to
optimize the particular paths. The rotation of the rotatable
perfusable culture chamber optimally controls collision
frequencies, collision intensities, and localization of the cells
in relation to other cells and also the limiting boundaries of the
rotatable perfusable culture chamber. If the cells are observed to
excessively distort inwards on the downward side and outwards on
the upwards side then RPM may preferably be increased. If the cells
are observed to centrifugate excessively to the outer walls then
the RPM may preferably be reduced. Not to be bound by theory, as
the operating limits are reached, in terms of high cell
sedimentation rates or high gravity strengths, the operator may be
unable to satisfy both of these conditions and may be forced to
accept degradation in performance as measured against the three
criteria above.
[0060] The cell sedimentation rate and the external gravitations
field place a lower limit on the fluid shear stress obtainable,
even within the operating range of the present invention, due to
gravitationally induced drift of the cells through the culture
medium of the three-dimensional TVEMF culture. Calculations and
measurements place this minimum fluid shear stress very nearly to
that resulting from the cells' terminal sedimentation velocity
(through the culture medium) for the external gravity field
strength. Centrifugal and coriolis induced motion [classical
angular kinematics provide the following equation relating the
Coriolis force to an object's mass (in), its velocity in a rotating
frame (v.sub.r) and the angular velocity of the rotating frame of
reference (): F.sub.Coriolis=-2 m (w.times.v.sub.r)] along with
secondary effects due to cell and culture medium interactions, act
to further degrade the fluid shear stress level as the cells, cell
aggregates, tissues, and tissue-like structures increase in
size.
[0061] Not to be bound by theory, but as the external gravity field
is reduced much denser and larger three-dimensional structures can
be obtained. In order to obtain the minimal fluid shear stress
level it is preferable that the chamber walls and internal
components be rotated at substantially the same rate as the culture
medium. Not to be bound by theory, but this minimizes the fluid
velocity gradient induced upon the three-dimensional TVEMF culture.
Preferably, selected levels of fluid shear stress may be introduced
to the three-dimensional TVEMF culture by differential rotation of
chamber components. It is advantageous to control the rate and size
of tissue formation in order to maintain the cell size (and
associated sedimentation rate) within a range for which the process
is able to satisfy the three criteria above. However, preferably,
the velocity gradient and resulting fluid shear stress may be
intentionally introduced and controlled for specific research
purposes such as studying the effects of shear stress on the
three-dimensional tissue. In addition, transient disruptions of the
stabilized culture environment are permitted and tolerated for,
among other reasons, logistical purposes during initial system
priming, sample acquisition, system maintenance, and culture
termination.
[0062] Rotating cells about an axis substantially perpendicular to
gravity can produce a variety of sedimentation rates, all of which
according to the present invention remain spatially localized in
distinct regions for extended periods of time ranging from seconds
(when sedimentation characteristics are large) to hours (when
sedimentation differences are small). Not to be bound by theory,
but this allows these cells, cell aggregates, tissues, and
tissue-like structures sufficient time to interact as necessary to
form multi-cellular structures and to associate with each other in
a three-dimensional TVEMF culture. Preferably, cells undergo
TVEMF-expansion preferably for at least 4 days, more preferably
from about 7 days to about 14 days, most preferably from about 7
days to about 10 days, even more preferably about 7 days.
Preferably, TVEMF-expansion may continue in a rotatable perfused
TVEMF-bioreactor of the present invention for up to 160 days, or
preferably at a rate of expansion that is about 1000 times the
original concentration of cells.
[0063] Rotatable perfusable culture chamber dimensions also
influence the path of cells in the three-dimensional TVEMF culture
of the present invention. A rotatable perfusable culture chamber
diameter is preferably chosen which has the appropriate volume,
preferably of from about 100 ml to about 3 L, for the intended
three-dimensional TVEMF culture and which will allow a sufficient
seeding density of cells. Not to be bound by theory, but the
outward cells drift due to centrifugal force is exaggerated at
higher rotatable perfusable culture chamber radii and for rapidly
sedimenting cells. Thus limiting the maximum radius of the
rotatable perfusable culture chamber as a function of the
sedimentation properties of the tissues anticipated in the final
three-dimensional TVEMF culture stages (when large tissues with
high rates of sedimentation have formed).
[0064] The path of the cells in the three-dimensional TVEMF culture
has been analytically calculated incorporating the cell motion
resulting from gravity, centrifugation, and coriolus effects. A
computer simulation of these governing equations allows the
operator to model the process and select parameters acceptable (or
optimal) for the particular planned three-dimensional TVEMF
culture. FIG. 6 shows the typical shape of the cell orbit as
observed from the external (non-rotating) reference frame. FIG. 7
is a graph of the radial deviation of a cell from the ideal
circular streamline plotted as a function of RPM (for a typical
cell sedimenting at 0.5 cm per second terminal velocity). This
graph (FIG. 7) shows the decreasing amplitude of the sinusoidally
varying radial cells deviation as induced by gravitational
sedimentation. FIG. 7 also shows increasing radial cells deviation
(per revolution) due to centrifugation as RPM is increased. These
opposing constraints influence carefully choosing the optimal RPM
to preferably minimize cell impact with, or accumulation at, the
chamber walls. A family of curves is generated which is
increasingly restrictive, in terms of workable RPM selections, as
the external gravity field strength is increased or the cell
sedimentation rate is increased. This family of curves, or
preferably the computer model which solves these governing orbit
equations, is preferably utilized to select the optimal RPM and
chamber dimensions for the TVEMF-expansion of cells of a given
sedimentation rate in a given external gravity field strength. Not
to be bound by theory, but as a typical three-dimensional TVEMF
culture is TVEMF expanded the tissues, cell aggregates, and
tissue-like structures increase in size and sedimentation rate, and
therefore, the rotation rate may preferably be adjusted to optimize
the same.
[0065] In the three-dimensional TVEMF culture, the cell orbit (FIG.
6) from the rotating reference frame of the culture medium is seen
to move in a nearly circular path under the influence of the
rotating gravity vector (FIG. 8). Not to be bound by theory, but
the two pseudo forces, coriolis and centrifugal, result from the
rotating (accelerated) reference frame and cause distortion of the
otherwise nearly circular path. Higher gravity levels and higher
cell sedimentation rates produce larger radius circular paths which
correspond to larger trajectory deviations from the ideal circular
orbit as seen in the non-rotating reference frame. In the rotating
reference frame it is thought, not to be bound by theory, that
cells of differing sedimentation rates will remain spatially
localized near each other for long periods of time with greatly
reduced net cumulative separation than if the gravity vector were
not rotated; the cells are sedimenting, but in a small circle (as
observed in the rotating reference frame). Thus, in operation the
present invention provides cells of differing sedimentation
properties with sufficient time to interact mechanically and
through soluble chemical signals. In operation, the present
invention provides for sedimentation rates of preferably from about
0 cm/second up to 10 cm/second.
[0066] Furthermore, the present invention provides that, in
operation, fresh or recycled culture medium may be moved within the
rotatable perfusable culture chamber preferably at a rate
sufficient to support metabolic gas exchange, nutrient delivery,
and metabolic waste product removal. This may slightly degrade the
otherwise quiescent three-dimensional TVEMF culture. In a preferred
embodiment, cells which sediment at about 0.5 cm per second, and
about 5 ml per minute culture medium perfused through a 500 ml
rotatable perfusable culture chamber, an average flow-speed of
about 0.001 cm per second is expected to result. Advantageously,
this is far slower than either gravitationally or centrifugally
induced cells motion. Preferably, the perfusion rate may be
increased as the cells' metabolic demand increases and a large
margin is available before significant fluid shear stress results
from the perfusion. It is preferable, therefore, to introduce a
mechanism for the support of preferred components including, but
not limited to, respiratory gas exchange, nutrient delivery, growth
factor delivery to the culture medium of the three-dimensional
TVEMF culture, and also a mechanism for metabolic waste product
removal in order to provide a long term three-dimensional TVEMF
culture able to support significant metabolic loads for periods of
hours to months.
[0067] In operation, the present invention not only provides for
high concentrations of cells that maintain their three-dimensional
geometry and cell-to-cell support but in addition, supplies TVEMF
to the cells in the three-dimensional TVEMF culture that may affect
some properties of cells during TVEMF-expansion. Without being
bound by the theory, for instance up-regulation of genes promoting
growth, or down regulation of genes preventing growth. In use, the
present invention provides that the cells in the three-dimensional
TVEMF culture are exposed to a TVEMF preferably of from about 0.05
gauss to about 6 gauss during TVEMF-expansion, more preferably of
from about 0.05 gauss to about 0.5 gauss, and most preferably about
0.5 gauss. The electromagnetic field is generated by a
TVEMF-source. In operation, the TVEMF-source of a rotatable
perfused TVEMF-bioreactor may preferably be rotatable with the
rotatable perfusable culture chamber, meaning about the same axis
as the rotatable perfusable culture chamber preferably in the same
direction. On the other hand, preferably the rotatable perfusable
culture chamber may be rotated in the opposite direction of the
TVEMF-source. Also, the TVEMF-source may preferably be fixed in
relation to a rotatable perfusable culture chamber of a rotatable
perfused TVEMF-bioreactor. The TVEMF-source may preferably be
integral with, meaning affixed to, the rotatable perfusable culture
chamber of a rotatable perfused TVEMF-bioreactor or may preferably
be adjacent to, preferably in close proximity to, more preferably
removably touching, the rotatable perfusable culture chamber of a
rotatable perfused TVEMF-bioreactor.
[0068] The time varying electromagnetic field is produced by a
varying electrical potential, preferably in the form of a delta
wave, more preferably a differential square wave, and most
preferably a square wave (following a Fourier curve), preferably
having a frequency of about 10 cycles per second. Preferably a
current of about 0.1 mA to about 1000 mA, and more preferably about
10 mA, produces a TVEMF extending at least several centimeters from
the conductive material. Typically, the range of frequency and
oscillating electromagnetic field strength is a parameter, which
may be selected for achieving the desired stimulation of particular
cells, and for providing the appropriate amount of up/down
regulation of genes, ultimately promoting TVEMF-expansion of
cells.
[0069] In addition to the qualitatively unique cells that are
produced by the operation of the present invention, not to be bound
by theory, an increased efficiency with respect to utilization of
the total rotatable perfusable culture chamber volume for cell and
tissue culture may be obtained due to the substantially uniform
homogeneous suspension achieved. Advantageously, therefore, the
present invention, in operation, provides an increased number of
cells or total tissue or tissue-like structure in the same
rotatable perfused TVEMF-bioreactor with less human resources. Many
cell types may be utilized in this process including, but not
limited to, mammalian, reptile, fish, yeast, and bacteria.
Fundamental cell and tissue biology research as well as clinical
applications requiring accurate in vitro models of in vivo cell
behavior are applications for which the present invention and
method of using the same provides an enhancement because, as
indicated above and throughout this application, TVEMF-expanded
cells and tissue of the present invention have essentially the same
three-dimensional geometry and cell-to-cell support and
cell-to-cell geometry as naturally-occurring, non-TVEMF-expanded
cells and tissue.
[0070] The method of the present invention provides these three
criteria above in a manner heretofore not obtained and optimizes a
three-dimensional TVEMF culture, and at the same time, provides the
TVEMF-expansion such that a sufficient rate of expansion (increase
in number per volume, diameter in reference to tissue, or
concentration) is detected in a sufficient amount of time. The
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned herein, as well as those
inherent therein. Without departing from the scope of the
invention, it is intended that all matter contained herein be
interpreted as illustrative and not limiting.
Operative Method
[0071] In operation, a rotatable perfused TVEMF-bioreactor
preferably having a rotatable perfusable culture chamber of from
100 ml to 3 L, is preferably first connected to a culture medium
flow loop including gas exchange membranes, a pump, and ports and
is then sterilized, preferably with ethylene oxide gas, and washed
with sterile phosphate buffered saline (PBS), watered, and aerated.
The rotatable perfusable culture chamber is completely filled with
the appropriate culture medium for the cells to be cultured, with
room only for any intended additional volumes of culture medium,
cells, and/or other preferred components of the culture medium of
the intended three-dimensional TVEMF culture. Preferrably a
controlled environment incubator completely surrounds the rotatable
perfused TVEMF-bioreactor and is preferably set for about 5%
CO.sub.2 and about 21% oxygen, and the temperature is preferably of
from about 26.degree. C. to about 41.degree. C., and more
preferably about 37.degree. C..+-.2.degree. C.
[0072] In a preferred embodiment, a culture medium flow loop is set
at a rate adequate to allow timely equilibration of the dissolved
gases with the external controlled incubator environment.
Initially, a stabilized culture environment is created in the
culture medium. The rotation may preferably begin at about 10
revolutions per minute (RPM), the slowest rate which produces a
microcarrier bead orbital trajectory in which the beads do not
accumulate appreciably at the chamber walls either by gravitational
induced settling or by rotationally induced centrifugation. All of
the adjustable rotating parts in the rotatable perfused
TVEMF-bioreactor are also set at the same rotation (preferably 10
RPM) in order to provide essentially no relative motion of the
three-dimensional TVEMF culture with respect to the chamber
internal surfaces. In this way, the rotatable perfused
TVEMF-bioreactor produces the minimal fluid velocity gradients and
fluid shear stresses in the three-dimensional TVEMF culture.
[0073] Cell attachment substrates are introduced either
simultaneously or sequentially with cells or tissue into the
rotatable perfusable culture chamber to give an appropriate
density, preferably 5 mg of cell attachment substrate per ml of
culture medium, and preferably the cell attachment substrate for
the anchorage dependent cells are microcarrier beads. The cells are
preferably injected into the stabilized culture environment,
preferably over a short period of time, preferably 2 minutes, so as
to minimize cell damage while passing through the delivery system.
The cells may preferably be injected into a rotatable perfusable
culture chamber with a syringe preferably into an injection port,
for instance that illustrated in FIGS. 4 and 5. The culture medium
is then rotated about a horizontal axis.
[0074] After injection of the cells is complete, the chamber outer
wall is quickly returned to initial rotation, preferably in less
than one (1) minute, preferably to match the angular rotational
rate of the rest of the system, more preferably 10 RPM, and thereby
return the shear stress to the minimal level obtainable for the
cells. During the initial loading and attachment phase, the cells
are allowed to equilibrate for a short period of time, preferably
of from 2 hours to 4 hours, more preferably for a time sufficient
for transient flows to dampen out.
[0075] In a preferred embodiment, the culture medium perfusion rate
is set to zero during cell or tissue loading and initial attachment
(cells attaching to the attachment substrate, preferably
microcarrier beads) so as to retain the cells within the rotatable
perfusable culture chamber as opposed to drawing them through the
filter and culture medium flow loop where severe cell damage would
occur. The absence of perfusion induced mixing, delivery of
nutrients and other preferred components of the culture medium,
waste product removal, and respiratory gas exchange during this
period is well tolerated due to the small total amount of initial
cellular metabolism and the brevity of this condition. Disruption
of the three-dimensional TVEMF culture could result from buoyant
gas interfering with the near solid body three-dimensional TVEMF
culture. Preferably, therefore, all free gas bubbles are purged,
preferably via a port, to promote minimal disruption to the
three-dimensional TVEMF culture.
[0076] Preferably after the initial loading and attachment phase
(preferably 2 to 4 hours) the culture medium flow loop is set at a
low flow speed (preferably 4.5 ml per minute) that does not
interfere with the initial three-dimensional assembly process. As
the TVEMF-expansion of the three-dimensional TVEMF culture
progresses the size and sedimentation rate of the assembled tissue
increases, the system rotational rates may be increased (increasing
in increments preferably of from about 1 to 2 RPM from about 10 to
30 RPM or more) in order to reduce the gravitationally induced
orbital distortion (from the ideal circular streamlines) of the now
increased diameter tissue pieces.
[0077] During TVEMF-expansion, the rotational speed of the
three-dimensional TVEMF culture in the rotatable perfusable culture
chamber may be assessed and adjusted so that the cell mixture
remains substantially at or about the horizontal axis. Increasing
the rotational speed is warranted to prevent wall impact, which is
detrimental to further three-dimensional growth of delicate
structure. For instance, an increase in the rotation is preferred
if the cells, tissue, or tissue-like structures in the
three-dimensional TVEMF culture fall excessively inward and
downward on the downward side of the rotation cycle and excessively
outward and insufficiently upward on the upward side of the
rotation cycle. Optimally, the user is advised to preferably select
a rotational rate that fosters minimal wall collision frequency and
intensity so as to maintain the three-dimensional geometry and
cell-to-cell support and cell-to-cell geometry of the cells,
tissue, or tissue-like structures. The preferred speed of the
present invention is of from about 5 to about 120 RPM, and more
preferably from about 10 to about 30 RPM.
[0078] The three-dimensional TVEMF culture may preferably be
visually assessed through the preferably transparent rotatable
perfusable culture chamber and manually adjusted. The assessment
and adjustment of the cell mixture may also be automated by a
sensor (for instance, a laser), which monitors the location of the
cell stem cells within a TVEMF-bioreactor. A sensor reading
indicating too much cell movement will automatically cause a
mechanism to adjust the rotational speed accordingly.
[0079] Furthermore, preferably after the initial loading and
attachment phase (2 to 4 hours) the TVEMF-source is turned on and
adjusted so that the TVEMF output generates the desired
electromagnetic field in the three-dimensional TVEMF culture in the
rotatable perfusable culture chamber, preferably in a range of from
0.05 gauss to 6 gauss, more preferably of from about 0.05 gauss to
about 0.5 gauss, and most preferably 0.5 gauss. The TVEMF can also
be applied to the three-dimensional TVEMF culture during the
initial loading and attachment phase. It is preferable that TVEMF
is supplied to the three-dimensional TVEMF culture for the length
of the culture time until the culture is terminated. Not to be
bound by theory, but TVEMF-expansion not only provides for high
concentrations of cells that maintain their three-dimensional
geometry and cell-to-cell support but TVEMF may affect some
properties of cells during TVEMF-expansion, for instance
up-regulation of genes promoting growth, or down regulation of
genes preventing growth.
[0080] The size of the wire coil, or preferably the annular wire
heater, of the TVEMF-source, and number of times it is wound, are
such that when a TVEMF, preferably in the form of a square wave,
preferably of from about 0.1 mA to about 1000 mA, more preferably
of from about 1 mA to about 10 mA, for instance 10 mA, is supplied
to the wire coil, a TVEMF, preferably of from about 0.05 gauss to
about 6 gauss, and more preferably of from about 0.05 to about 0.5
gauss, and most preferably 0.5 gauss, is generated within the
three-dimensional TVEMF culture in the rotatable perfusable culture
chamber of the rotatable perfused TVEMF-bioreactor. Preferably, the
square wave has a frequency of about 2 to about 25 cycles/second,
more preferably about 5 to about 20 cycles/second, and for example
about 10 cycles/second. However, these parameters are not meant to
be limiting to the TVEMF of the present invention, as such may vary
based on other aspects of this invention. TVEMF may be measured for
instance by standard equipment such as an EN131 Cell Sensor Gauss
Meter.
[0081] The rapid cell and tissue expansion and increasing total
metabolic demand may necessitate additional intermittent addition
of preferable components enriching the culture medium in the
three-dimensional TVEMF culture including, but not limited to,
nutrients, fresh culture medium, growth factors, hormones, and
cytokines. This addition is preferably increased as necessary to
maintain glucose and other nutrient levels. During the rapid cell
and tissue expansion, spent culture medium comprising waste may
preferably be removed. The three-dimensional TVEMF culture may be
allowed to progress beyond the point at which it is possible to
select excellent cells orbits; at a point when gravity has
introduced constraints which somewhat degrade performance in terms
of a low shear three-dimensional TVEMF culture.
[0082] In a preferred embodiment of the present invention,
preferably every 15 minutes during the total culture period the
inner filter assembly is stopped and started at 15-second
intervals, preferably for 1 minute, at least in order to clear
cells from the inner filter assembly surfaces. This prevents
accumulation of substrates, cells, and debris on the filter. In
addition, samples of the cells in the three-dimensional TVEMF
culture may be collected as desired. If the samples are to be
collected via a syringe, the chamber outer wall may be temporarily
stopped to allow practical handling.
Example I
Three-Dimensional Rat Bone Cell Culture
Preparation
[0083] A 100 ml rotatable perfused TVEMF-bioreactor, illustrated in
the preferred embodiment of FIG. 4, was prepared by washing with
detergent and germicidal disinfectant solution (Roccal II) at the
recommended concentration for disinfection and cleaning followed by
copious rinsing and soaking with high quality deionized water. The
rotatable perfused TVEMF-bioreactor was sterilized by autoclaving
then rinsed once with culture medium.
Inoculation
[0084] The rotatable perfused TVEMF-bioreactor was filled with
culture medium consisting of minimum essential medium (MEM) with
Earle's salts, growth supplements, antibiotics and 10% fetal bovine
serum. After equilibration for one (1) hour in a CO.sub.2
incubator, at 5% CO.sub.2 environment at 37.degree. C., the
substrate consisting of collagen coated dextian polymer, Cytodex 3
microcarrier beads (Pharmacia Fine Chemicals, Uppsala, Sweden) were
suspended in a small volume of culture medium and loaded into the
rotatable perfusable culture chamber of the rotatable perfused
TVEMF-bioreactor. An empty syringe attached to one of the sampling
ports functioned as a compliant volume reservoir during inoculation
to receive the displaced media. The final bead concentration was 5
mg/ml of chamber volume.
[0085] A volume of culture medium was injected to completely fill
the chamber. All air bubbles were removed using a syringe attached
to a sample port on the chamber. The rotatable perfusable culture
chamber of the rotatable perfused TVEMF-bioreactor containing
suspended beads was equilibrated for about 30 minutes.
[0086] Mono-dispersed primary rat osteoblast calvarium bone cells
were suspended in a small volume of the same culture medium already
in the rotatable perfusable culture chamber at a concentration of
5.times.10.sup.5 cells/ml. 50 ml at a concentration of
5.times.10.sup.5 cells/ml were then inoculated into the rotatable
perfusable culture chamber by injection through a sample port. The
cell seeding density was approximately 10 cells/bead. A second
syringe was attached to another port and served as a compliant
volume reservoir for the chamber. The motor was switched on and the
rotatable perfused TVEMF bioreactor was rotated at a rate of
approximately 16 RPM. The TVEMF source was also turned on to a
gauss level of 0.05 gauss in a square wave form (following a
Fourier curve).
Cell Attachment and Three Dimensional Growth
[0087] At 24 hours, the rotation of the rotatable perfused
TVEMF-bioreactor was stopped, a sample was collected through a
syringe, and the rotation rate was again increased to the rate that
was applied before the collection, approximately 16 RPM. The sample
was analyzed under a microscope (400.times. magnification).
Microscopic observation showed that the cells were well attached
and flattened on the surface of the beads. The beads and cells were
not associated into higher order structures at this point. Very
little orbital path distortion or centrifugation was observed. The
medium was changed by simultaneously removing medium with a syringe
and adding a volume of the same with another to remove non-viable
floating cells. On day three, the medium was changed again, as
above, to assure nutrient supply to the cells. At day four cells
were in good condition. To replenish nutrients yet retain growth
factors secreted by the cells, three-fourths of the medium was
removed and the same volume of fresh medium was added by using two
syringes simultaneously as above. The same procedure was repeated
on day 5.
[0088] On day 5, a sample of the three-dimensional TVEMF culture
was again collected and microscopic observation of samples showed
rounded assemblies of beads that were larger than seen on the
previous day. Also noted were cells spanning spaces between beads.
The airflow into the device was turned up to provide more oxygen to
the rapidly growing culture. At this point the assemblies were
observed to fall radially inwards on the "down" side and outwards
on the "upside".
[0089] On the 6th day the culture was transferred to a larger 250
ml volume rotatable perfused TVEMF-bioreactor, as in FIG. 5. On day
8, ordered assemblies of beads were very large (1-2 mm) with 8 to
15 beads in the assemblies and more three-dimensional structure was
noted. The medium was changed at this time. At this point some
accumulation of the tissue assemblies was occurring at the outer
chamber wall. This centrifugation effect was quite gentle.
[0090] The cells were maintained in the three dimensional structure
for 17 days with additional medium changes on days 9, 10 and 12 and
additions of glucose on days 11, 15 and 16. The results were
visually assessed by both the naked eye, and also via a microscope
(400.times. magnification). There was no evidence of mechanical
damage and the size of the cell/bead assemblies did not exceed the
ability of the device to suspend the cells. The assemblies were of
1 to 2 mm in size and consisted of cells of mixed morphology which
may be the indicia of beginning differentiation. The run was
terminated by choice of the investigators. Prior to the
TVEMF-expansion, the sample had 5.times.10.sup.5 cells/ml. After
the TVEMF-expansion, the sample had 4.2.times.10.sup.6
cells/ml.
Example 2
Formation of Artificial Tissue in Suspension
Preparation
[0091] A 500 ml rotatable perfused TVEMF-bioreactor consisting of a
500 ml cell rotatable perfusable culture chamber, a hollow fiber
oxygenator, a prototype diaphragm pump, an in-line pH sensor,
sample ports and a peristaltic pump for infusion of fresh medium
were assembled, sterilized by ethylene oxide (ETO) and aerated for
two days. The chamber was then loaded with phosphate buffered
saline (PBS) to rinse and remove residual ETO. During this step, a
leak was discovered in the oxygenator and unit was replaced using
sterile techniques. The system was then loaded with culture medium
comprising minimum essential medium (MEM) with Earle's salts,
growth supplements, antibiotics and 10% fetal bovine serum and
placed in the CO.sub.2 incubator at a 5% CO.sub.2 environment and
37.degree. C. After remaining sterile for at least two days, the
rotatable perfusable culture chamber was loaded with cells and
substrate as described below.
Inoculation
[0092] Cytodex 3 microcarrier beads (Pharmacia Fine Chemicals,
Uppsala, Sweden) were reconstituted according to standard
laboratory procedures, suspended in Microcarrier Medium (MM),
containing 20% fetal calf serum, 100 units of penicillin/ml and 100
ug of streptomycin/ml, loaded into a 50 ml syringe and injected
into the chamber. The bead density in the chamber was 5 mg/ml of
chamber volume.
[0093] The system was then loaded with Baby Hamster Kidney (BHK21)
cells, at 56 passages. To achieve this, ampules of frozen stock
BHK21 cells were thawed. The cells were suspended in 50 ml of
Microcarrier (MM) at a concentration of 0.75.times.10.sup.6
cells/ml and all 50 ml were injected into the rotatable perfusable
culture chamber through a 20 ml syringe (2.5 injections) The final
cell seeding density was approximately 6 cells/bead. Cells were
loaded into the chamber at 9:30 AM.
[0094] The system parameters were as follows. The constant volume
diaphragm pump was timed to circulate the culture medium at 4.5
ml/min. The pump rate was turned up to 20 ml/min four days after
addition of cells and beads in order to increase the oxygen
delivery to the reactor chamber. The pump system delivered 0.7 ml
every two seconds. The rotation rate of the chamber and the blade
members were set at 15 to 20 RPM and remained there during the
test. The filter assembly rotation rate was 25 to 30 RPM. This
produced a very low turbulence environment resulting in a cell/bead
suspension upon introduction of those cells. The TVEMF source was
also turned on to a gauss level of 0.05 gauss in a square wave form
(following a Fourier curve).
Cell Attachment and Three-Dimensional Growth
[0095] In order to assess the rate of attachment of cells to the
substrate beads, samples of the three-dimensional TVEMF culture,
containing cell-bead assemblies, were removed via a syringe for
cell counts and microscopic observation at 2, 4, and 6 hours after
initially loading the chamber with cells. Many of the cells
attached to the beads within two hours and flattened on the surface
of the beads, which is an essential state preliminary to growth of
the cells. Early in this experiment, microscopic observation showed
that some of the mono-dispersed cells clumped in groups of 10 to 30
cells. At 4 hours, these clumps of cells had attached to beads and
were flattened on the surfaces. No orbital distortion or
centrifugation effects were visible at this point.
[0096] At 24 hours some beads were covered with cells but there
were also many floating cells. At this time, fresh medium was
perfusion into the chamber through a port or passageway. Four hours
later, a sample was collected and upon visual microscopic
observation it appeared that almost all of the cells had attached
to beads and there was no evidence of cell-cell aggregates as seen
earlier. The poor appearance at 24 hours was probably due to
toxicity from the new oxygenator used to replace the original one
that leaked during the set up.
[0097] At 48 hours, the cells were visually assessed through the
rotatable perfusable culture chamber and it was determined that the
cell growth rate had increased rapidly. Glucose, glutamine
(nutrients) and sodium hydroxide for pH control were added on day
four to compensate for cell metabolism and depletion of
nutrients.
[0098] On day 5 aggregates of cells and beads were visually noted
and the medium was changed to minimum essential medium (MEM) with
Earle's salts, growth supplements, antibiotics and containing 2%
fetal calf serum, to slow cell metabolism and growth. The cells
continued to grow, however, and reached a maximum density of 148
cells/bead at day 7. Very large and uniformed assemblies of about 1
to 2 mm formed and did not disaggregate during the remainder of the
test. Ordered uniform assemblies of 8 to 10 beads formed in
three-dimensional arrays over which the cells grew into a smooth
membrane-like configuration of elongated fibroblastoid morphology.
Multiple layers of these cells were apparent by microscope
examination both on the surface and in the inter-bead spaces. The
cells were placed in ordered layers with cell membranes immediately
adjacent to each other. The test was terminated on day 10. The size
of most of the tissue-like aggregates of cells on beads did not
exceed the limits of the rotatable perfusable culture chamber of
the rotatable perfused TVEMF-bioreactor to freely suspend the
aggregates in a quiescent three-dimensional TVEMF culture,
unrestricted by internal boundaries. Some very large assemblies, 3
to 8 mm, were observed. These rapidly sedimenting tissues were
observed to exhibit grossly distorted orbital paths and
centrifugation to the outer chamber walls. High-energy impacts and
vigorous "rolling" effects were observed. These were considered
beyond the process capacity to retain the quiescent, low shear,
three-dimensional TVEMF culture. Prior to the TVEMF-expansion, the
sample had 0.75.times.10.sup.6 cells/ml. After the TVEMF-expansion,
the sample had 5.4.times.10.sup.6 cells/ml.
* * * * *